Recombinant Vaccine Displaying the Loop-Neutralizing Determinant from Protective Antigen Completely Protects Rabbits from Experimental Inhalation Anthrax

Abstract

We previously showed that a multiple antigenic peptide (MAP) vaccine displaying amino acids (aa) 304 to 319 from the 2␤2-2␤3 loop of protective antigen was capable of protecting rabbits from an aerosolized spore challenge with Bacillus anthracis Ames strain. Antibodies to this sequence, referred to as the loop-neutralizing determinant (LND), are highly potent at neutralizing lethal toxin yet are virtually absent in rabbit and human protective antigen (PA) antiserum. While the MAP vaccine was protective against anthrax, it contains a single heterologous helper T cell epitope which may be suboptimal for stimulating an outbred human population. We therefore engineered a recombinant vaccine (Rec-LND) containing two tandemly repeated copies of the LND fused to maltose binding protein, with enhanced immunogenicity resulting from the p38/P4 helper T cell epitope from Schistosoma mansoni. Rec-LND was found to be highly immunogenic in four major histocompatibility complex (MHC)-diverse strains of mice. All (7/7) rabbits immunized with Rec-LND developed high-titer antibody, 6 out of 7 developed neutralizing antibody, and all rabbits were protected from an aerosolized spore challenge of 193 50% lethal doses (LD 50 ) of the B. anthracis Ames strain. Survivor serum from Rec-LND-immunized rabbits revealed significantly increased neutralization titers and specific activity compared to prechallenge levels yet lacked PA or lethal factor (LF) antigenemia. Control rabbits immunized with PA, which were also completely protected, appeared sterilely immune, exhibiting significant declines in neutralization titer and specific activity compared to prechallenge levels. We conclude that Rec-LND may represent a prototype anthrax vaccine for use alone or potentially combined with PA-containing vaccines.

Anthrax Pathogenesis

“…Recently advances in enhancement of the immune response through cofactors targeting TLR signaling have been reported as well (219). A new vaccine using recombinant PA has not yet been approved; however, several, including those utilizing mutant PA variants with less toxicity and higher immunogenicity, are in development or clinical trials (17,25,68,86,118,136,188,189,277).…”

Anthrax vaccine recipients lack antibody against the loop neutralizing determinant: A protective neutralizing epitope from Bacillus anthracis protective antigen

“…We have previously shown that immunization with epitope-focused immunogens using either research adjuvants like Freund's or AlOH-containing human-use adjuvants can elicit Ab specific for a linear determinant in the 2β2-2β3 loop of PA which can mediate protection of rabbits from aerosolized spore challenge with B. anthracis Ames strain [10-12]. This epitope, referred to as the loop neutralizing determinant (LND), appears to be a functionally critical target for Ab, as relatively low serum titers of LND-specific Ab are capable of protecting rabbits from high dose aerosol challenge.…”

Epitope-focused peptide immunogens in human use adjuvants protect rabbits from experimental inhalation anthrax

“…We have previously shown that immunization with epitope-focused immunogens can elicit Ab specific for a linear determinant in the 2β2–2β3 loop of protective antigen (PA) which can mediate protection of rabbits from spore challenge with B. anthracis Ames strain [7,8]. This protective neutralizing epitope, referred to as the loop neutralizing determinant (LND), is found within a segment of PA critical to the translocation of edema and lethal factor into cells [9–11].…”